Structure /Function of Phospholamban in Heart

描述（由申请人提供）：本研究的长期目标是阐明受磷蛋白（PLB）抑制心脏肌浆网（SR）中钙泵（SERCA 2a亚型）活性的分子和生物物理机制。PLB是心脏SR中的五聚体磷蛋白，其由五个相同的单体组成。以前，我们证明了PLB单体负责结合SERCA 2a并抑制它，现在，我们建议定位PLB单体和SERCA 2a之间的结合相互作用位点，导致酶抑制，并确定分子相互作用如何受到关键的变构调节剂，包括Ca浓度，核苷酸和磷酸化。重点将放在识别氨基酸的抑制复合物，直接相互作用，利用我们新开发的化学交联方法。在目的1中，我们将进行PLB的Cys扫描诱变以定位沿着其一级结构的与SERCA 2a的内源性Cys残基交联的不同位点沿着。SERCA 2a的交联Cys残基将通过蛋白质纯化/肽测序直接鉴定。在目标2中，将定位交联至PLB的不同位点的SERCA 2a的Lys残基。通过使用交联剂作为分子标尺并结合目标1和2的结果，我们将开发PLB单体和SERCA 2a之间形成的结合复合物的精确3-D模型。在目的3中，将研究Ca浓度、核苷酸和抑制剂毒胡萝卜素对PLB与SERCA 2a交联的影响。检验的假设是PLB仅结合SERCA 2a的无Ca形式（E2），但仅结合ATP或ADP的E2状态。在目的4中，我们将确定Ca如何缓解PLB对SERCA 2a的抑制。我们假设PLB优先结合E2，拮抗Ca结合SERCA 2a，Ca优先结合E1，拮抗PLB结合SERCA 2a。将鉴定负责使PLB与泵解离的SERCA 2a的Ca结合位点，并将定量PLB对SERCA 2a的Ca结合亲和力的影响。在目标5中，我们将确定蛋白激酶对PLB的磷酸化如何减轻PLB抑制。所测试的假设是PLB的磷酸化直接使其与SERCA 2a解离。在这里，我们还将确定PLB的两个磷酸化残基Ser 16和Thr 17是否直接与SERCA 2a相互作用以帮助酶抑制。PLB是心肌收缩动力学的关键调节因子。通过定义其对Ca泵的分子作用机制，将产生对PLB调节心跳强度的新见解，这可能最终导致设计治疗心力衰竭的新药。

DESCRIPTION (provided by applicant): The long term goal of this research is to elucidate the molecular and biophysical mechanism by which phospholamban (PLB) inhibits the activity of the Ca pump (SERCA2a isoform) in cardiac sarcoplasmic reticulum (SR). PLB is a pentameric phosphoprotein in cardiac SR, which is composed of five identical monomers. Previously, we demonstrated the PLB monomer is responsible for binding to SERCA2a and inhibiting it. Now, we propose to localize the binding-interaction sites between the PLB monomer and SERCA2a that lead to enzyme inhibition, and determine how the molecular interaction is regulated by key allosteric modulators including Ca concentration, nucleotides, and phosphorylation. Emphasis will be placed upon identifying amino acids in the inhibitory complex that interact directly, taking advantage of our newly developed chemical cross-linking method. In Aim 1, we will perform Cys-scanning mutagenesis of PLB to localize distinct sites along its primary structure that cross-link to endogenous Cys residues of SERCA2a. The cross-linked Cys residues of SERCA2a will be directly identified by protein purification/peptide sequencing. In Aim 2, Lys residues of SERCA2a that cross-link to distinct sites of PLB will be localized. By use of crosslinking agents as molecular rulers and combining results from Aims 1 and 2, we will develop an accurate 3-D model of the binding-complex formed between the PLB monomer and SERCA2a. In Aim 3, the effects of Ca concentration, nucleotides, and the inhibitor thapsigargin on cross-linking of PLB to SERCA2a will be investigated. The hypothesis tested is that PLB binds exclusively to the Ca-free form (E2) of SERCA2a, but only that E2 state that has bound ATP or ADP. In Aim 4, we will determine how Ca relieves PLB inhibition of SERCA2a. We hypothesize that PLB binds preferentially to E2, antagonizing Ca binding to SERCA2a, and that Ca binds preferentially to El, antagonizing PLB binding to SERCA2a. The Ca-binding site of SERCA2a responsible for dissociating PLB from the pump will be identified, and the effect of PLB on the Ca-binding affinity of SERCA2a will be quantified. In Aim 5, we will determine how phosphorylation of PLB by protein kinases relieves PLB inhibition. The hypothesis tested is that phosphorylation of PLB directly dissociates it from SERCA2a. Here we will also determine if the two phosphorylated residues of PLB, Ser 16 and Thr 17, interact directly with SERCA2a to aid in enzyme inhibition. PLB is a key regulator of myocardial contractile dynamics. By defining its molecular mechanism of action on the Ca pump, new insights on PLB regulation of the strength of the heartbeat will result, that may ultimately lead to the design of new drugs to treat heart failure.